Supplementary Figure 5 from High Aldehyde Dehydrogenase Activity Identifies Tumor-Initiating and Metastasis-Initiating Cells in Human Prostate Cancer

Transformed epithelial cells can activate programs of epithelial plasticity and switch from a sessile, epithelial phenotype to a motile, mesenchymal phenotype. This process is linked to the acquisition of an invasive phenotype and the formation of distant metastases. The development of compounds that block the acquisition of an invasive phenotype or revert the invasive mesenchymal phenotype into a more differentiated epithelial phenotype represent a promising anticancer strategy. In a high-throughput assay based on E-cadherin (re)induction and the inhibition of tumor cell invasion, 44,475 low molecular weight (LMW) compounds were screened. The screening resulted in the identification of candidate compounds from the PROAM02 class. Selected LMW compounds activated E-cadherin promoter activity and inhibited cancer cell invasion in multiple metastatic human cancer cell lines. The intraperitoneal administration of selected LMW compounds reduced the tumor burden in human prostate and breast cancer in vivo mouse models. Moreover, selected LMW compounds decreased the intra-bone growth of xenografted human prostate cancer cells. This study describes the identification of the PROAM02 class of small molecules that can be exploited to reduce cancer cell invasion and metastases. Further clinical evaluation of selected candidate inhibitors is warranted to address their safety, bioavailability and antitumor efficacy in the management of patients with aggressive cancers.

Current treatments of advanced prostate cancer only marginally increase overall survival and can be regarded as predominantly palliative. Hence, there is an urgent need for novel therapeutic strategies for the treatment of primary tumors and, more importantly perhaps, for the prevention of tumor progression and metastasis formation. Clinically relevant preclinical models are therefore urgently needed. An ideal, clinically relevant preclinical model would mimic the genetic and phenotypic changes that occur at the different stages of human prostate cancer progression and subsequent metastasis. In this chapter, transplantable xenograft prostate cancer models are described, in which human prostate cancer cells are transplanted into host animals (e.g., immune-deficient mice). Cancer cells can be administered to the small laboratory animals in various ways, including inoculation of the prostate tumor cells subcutaneously, at the anatomical site of origin (orthotopically), or at the metastatic site. In addition, we describe imaging methods suitable for small laboratory animals with emphasis on optical imaging (bioluminescence and fluorescence).

Transcription factors have an important role in cancer but are difficult targets for the development of tumour therapies. These factors include the Ets family, and in this study Elk3 that is activated by Ras oncogene /Erk signalling, and is involved in angiogenesis, malignant progression and epithelial-mesenchymal type processes. We previously described the identification and in-vitro characterisation of an inhibitor of Ras / Erk activation of Elk3 that also affects microtubules, XRP44X. We now report an initial characterisation of the effects of XRP44X in-vivo on tumour growth and metastasis in three preclinical models mouse models, subcutaneous xenografts, intra-cardiac injection-bone metastasis and the TRAMP transgenic mouse model of prostate cancer progression. XRP44X inhibits tumour growth and metastasis, with limited toxicity. Tumours from XRP44X-treated animals have decreased expression of genes containing Elk3-like binding motifs in their promoters, Elk3 protein and phosphorylated Elk3, suggesting that perhaps XRP44X acts in part by inhibiting the activity of Elk3. Further studies are now warranted to develop XRP44X for tumour therapy.

Background The inflammatory tumor microenvironment, and more specifically the tumor‐associated macrophages, plays an essential role in the development and progression of prostate cancer towards metastatic bone disease. Tumors are often characterized by a leaky vasculature, which ‐ combined with the prolonged circulation kinetics of liposomes ‐ leads to efficient tumor localization of these drug carriers, via the so‐called enhanced permeability and retention (EPR) ‐effect. In this study, we evaluated the utility of targeted, liposomal drug delivery of the glucocorticoid dexamethasone in a model of prostate cancer bone metastases. Methods Tumor‐bearing Balb‐c nu/nu mice were treated intravenously with 0.2–1.0–5.0 mg/kg/week free‐ and liposomal DEX for 3–4 weeks and tumor growth was monitored by bioluminescent imaging. Results Intravenously administered liposomes localize efficiently to bone metastases in vivo and treatment of established bone metastases with (liposomal) dexamethasone resulted in a significant inhibition of tumor growth up to 26 days after initiation of treatment. Furthermore, 1.0 mg/kg liposomal dexamethasone significantly outperformed 1.0 mg/kg free dexamethasone, and was found to be well‐tolerated at clinically‐relevant dosages that display potent anti‐tumor efficacy. Conclusions Liposomal delivery of the glucocorticoid dexamethasone inhibits the growth of malignant bone lesions. We believe that liposomal encapsulation of dexamethasone offers a promising new treatment option for advanced, metastatic prostate cancer which supports further clinical evaluation. Prostate 75: 815–824, 2015. © 2015 The Authors. The Prostate , published by Wiley Periodicals, Inc.

Low survival rates of metastatic cancers emphasize the need for a drug that can prevent and/or treat metastatic cancer. αv integrins are involved in essential processes for tumor growth and metastasis and targeting of αv integrins has been shown to decrease angiogenesis, tumor growth and metastasis. In this study, the role of αv integrin and its potential as a drug target in bladder cancer was investigated. Treatment with an αv integrin antagonist as well as knockdown of αv integrin in the bladder carcinoma cell lines, resulted in reduced malignancy in vitro, as illustrated by decreased proliferative, migratory and clonogenic capacity. The CDH1/CDH2 ratio increased, indicating a shift towards a more epithelial phenotype. This shift appeared to be associated with downregulation of EMT-inducing transcription factors including SNAI2. The expression levels of the self-renewal genes NANOG and BMI1 decreased as well as the number of cells with high Aldehyde Dehydrogenase activity. In addition, self-renewal ability decreased as measured with the urosphere assay. In line with these observations, knockdown or treatment of αv integrins resulted in decreased metastatic growth in preclinical in vivo models as assessed by bioluminescence imaging. In conclusion, we show that αv integrins are involved in migration, EMT and maintenance of Aldehyde Dehydrogenase activity in bladder cancer cells. Targeting of αv integrins might be a promising approach for treatment and/or prevention of metastatic bladder cancer.

Invasive ductal carcinoma (IDC) and invasive lobular carcinoma (ILC) are the most frequently occurring histological subtypes of breast cancer, accounting for 80-90% and 10-15% of the total cases, respectively. At the time of diagnosis and surgical resection of the primary tumour, most patients do not have clinical signs of metastases, but bone micrometastases may already be present. Our aim was to develop a novel preclinical ILC model of spontaneous bone micrometastasis. We used murine invasive lobular breast carcinoma cells (KEP) that were generated by targeted deletion of E-cadherin and p53 in a conditional K14cre;Cdh1((F/F));Trp53((F/F)) mouse model of de novo mammary tumour formation. After surgical resection of the growing orthotopically implanted KEP cells, distant metastases were formed. In contrast to other orthotopic breast cancer models, KEP cells readily formed skeletal metastases with minimal lung involvement. Continuous treatment with SD-208 (60mg/kg per day), an orally available TGF receptor I kinase inhibitor, increased the tumour growth at the primary site and increased the number of distant metastases. Furthermore, when SD-208 treatment was started after surgical resection of the orthotopic tumour, increased bone colonisation was also observed (versus vehicle). Both our in vitro and in vivo data show that SD-208 treatment reduced TGF signalling, inhibited apoptosis, and increased proliferation. In conclusion, we have demonstrated that orthotopic implantation of murine ILC cells represent a new breast cancer model of minimal residual disease in vivo, which comprises key steps of the metastatic cascade. The cancer cells are sensitive to the anti-tumour effects of TGF. Our in vivo model is ideally suited for functional studies and evaluation of new pharmacological intervention strategies that may target one or more steps along the metastatic cascade of events. (c) 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cancer cells with stem or progenitor properties play a pivotal role in the initiation, recurrence and metastatic potential of solid tumors, including those of the human prostate. Cancer stem cells are generally more resistant to conventional therapies thus requiring the characterization of key pathways involved in the formation and/or maintenance of this malignant cellular subpopulation. To this end, we identified Glycogen Synthase Kinase-3β (GSK-3β) as a crucial kinase for the maintenance of prostate cancer stem/progenitor-like cells and pharmacologic inhibition of GSK-3β dramatically decreased the size of this cellular subpopulation. This was paralleled by impaired clonogenicity, decreased migratory potential and dramatic morphological changes. In line with our in vitro observations, treatment with a GSK-3β inhibitor leads to a complete loss of tumorigenicity and a decrease in metastatic potential in preclinical in vivo models. These observed anti-tumor effects appear to be largely Wnt-independent as simultaneous Wnt inhibition does not reverse the observed antitumor effects of GSK-3β blockage. We found that GSK-3β activity is linked to cytoskeletal protein F-actin and inhibition of GSK-3β leads to disturbance of F-actin polymerization. This may underlie the dramatic effects of GSK-3β inhibition on prostate cancer migration. Furthermore, GSK-3β inhibition led to strongly decreased expression of several integrin types including the cancer stem cell-associated α2β1 integrin. Taken together, our mechanistic observations highlight the importance of GSK-3β activity in prostate cancer stemness and may facilitate the development of novel therapy for advanced prostate cancer.

High aldehyde dehydrogenase (ALDH) activity can be used to identify tumor-initiating and metastasis-initiating cells in various human carcinomas, including prostate cancer. To date, the functional importance of ALDH enzymes in prostate carcinogenesis, progression and metastasis has remained elusive. Previously we identified strong expression of ALDH7A1 in human prostate cancer cell lines, primary tumors and matched bone metastases. In this study, we evaluated whether ALDH7A1 is required for the acquisition of a metastatic stem/progenitor cell phenotype in human prostate cancer. Knockdown of ALDH7A1 expression resulted in a decrease of the α2(hi)/αv(hi)/CD44(+) stem/progenitor cell subpopulation in the human prostate cancer cell line PC-3M-Pro4. In addition, ALDH7A1 knockdown significantly inhibited the clonogenic and migratory ability of human prostate cancer cells in vitro. Furthermore, a number of genes/factors involved in migration, invasion and metastasis were affected including transcription factors (snail, snail2, and twist) and osteopontin, an ECM molecule involved in metastasis. Knockdown of ALDH7A1 resulted in decreased intra-bone growth and inhibited experimentally induced (bone) metastasis, while intra-prostatic growth was not affected. In line with these observations, evidence is presented that TGF-β, a key player in cancer invasiveness and bone metastasis, strongly induced ALDH activity while BMP7 (an antagonist of TGF-β signaling) down-regulated ALDH activity. Our findings show, for the first time, that the ALDH7A1 enzyme is functionally involved in the formation of bone metastases and that the effect appeared dependent on the microenvironment, i.e., bone versus prostate.

Background: Bladder cancer is the fifth most common malignancy in the Western world and the second most frequently diagnosed genitourinary tumor. In the majority of cases, death from bladder cancer results from metastatic disease. Understanding the multistep process of carcinogenesis and metastasis in urothelial cancers is pivotal to the development of new therapeutic strategies. Molecular imaging of cancer growth and metastasis in preclinical models provides the essential link between cell-based experiments and clinical translation.Objective: Develop preclinical models for sensitive bladder cancer cell tracking during tumor progression and metastasis.Design, setting, and participants: A human transitional cell carcinoma UM-UC-3 cell line was generated that stably expresses luciferase 2 (UM-UC-3luc2), a mammalian codon-optimized firefly luciferase with superior expression. Preclinical models were developed with human UM-UC-3luc2 cells xenografted into the bladder (orthotopic model with metastases) or inoculated into the left cardiac ventricle (bone metastasis model) of immunocompromised mice.Measurements: Noninvasive, sensitive bioluminescent imaging of human firefly luciferase 2-positive bladder cancer in mice using the IVIS100 imaging system.Results and limitations: In the orthotopic model (intravesical inoculation), tumor growth could be followed directly after inoculation of UM-UC-3luc2 cells. Importantly, micrometastatic lesions originating from orthotopically implanted cancer cells could be detected in the locoregional lymph nodes and in distant organs. In addition, the superior bioluminescent indicator firefly luciferase 2 allows the detection and monitoring of micrometastatic lesions in real time after intracardiac inoculation of human bladder cancer cells in mice. The main disadvantage is the lack of T-cell immunity in the preclinical models.Conclusions: The new bioluminescence-based preclinical bladder cancer models enable superior, noninvasive, and real-time tracking of cancer cells, tumor progression, and micrometastasis. Because of the significant improvement in detection of small cell numbers, the presented models are ideally suited for functional studies dealing with minimal residual disease as well as real-time imaging of drug response. Crown Copyright (C) 2011 Published by Elsevier B. V. on behalf of European Association of Urology. All rights reserved.

Integrins participate in multiple cellular processes, including cell adhesion, migration, proliferation, survival, and the activation of growth factor receptors. Recent studies have shown that expression of αv integrins is elevated in the prostate cancer stem/progenitor cell subpopulation compared with more differentiated, committed precursors. Here, we examine the functional role of αv integrin receptor expression in the acquisition of a metastatic stem/progenitor phenotype in human prostate cancer. Stable knockdown of αv integrins expression in PC-3M-Pro4 prostate cancer cells coincided with a significant decrease of prostate cancer stem/progenitor cell characteristics (α2 integrin, CD44, and ALDH(hi)) and decreased expression of invasion-associated genes Snail, Snail2, and Twist. Consistent with these observations, αv-knockdown strongly inhibited the clonogenic and migratory potentials of human prostate cancer cells in vitro and significantly decreased tumorigenicity and metastatic ability in preclinical models of orthotopic growth and bone metastasis. Our data indicate that integrin αv expression is functionally involved in the maintenance of a highly migratory, mesenchymal cellular phenotype as well as the acquisition of a stem/progenitor phenotype in human prostate cancer cells with metastasis-initiating capacity.

Acquisition of an invasive phenotype by cancer cells is a requirement for bone metastasis. Transformed epithelial cells can switch to a motile, mesenchymal phenotype by epithelial-mesenchymal transition (EMT). Recently, it has been shown that EMT is functionally linked to prostate cancer stem cells, which are not only critically involved in prostate cancer maintenance but also in bone metastasis. We showed that treatment with the non-peptide αv-integrin antagonist GLPG0187 dose-dependently increased the E-cadherin/vimentin ratio, rendering the cells a more epithelial, sessile phenotype. In addition, GLPG0187 dose-dependently diminished the size of the aldehyde dehydrogenase high subpopulation of prostate cancer cells, suggesting that αv-integrin plays an important role in maintaining the prostate cancer stem/progenitor pool. Our data show that GLPG0187 is a potent inhibitor of osteoclastic bone resorption and angiogenesis in vitro and in vivo. Real-time bioluminescent imaging in preclinical models of prostate cancer demonstrated that blocking αv-integrins by GLPG0187 markedly reduced their metastatic tumor growth according to preventive and curative protocols. Bone tumor burden was significantly lower in the preventive protocol. In addition, the number of bone metastases/mouse was significantly inhibited. In the curative protocol, the progression of bone metastases and the formation of new bone metastases during the treatment period was significantly inhibited. In conclusion, we demonstrate that targeting of integrins by GLPG0187 can inhibit the de novo formation and progression of bone metastases in prostate cancer by antitumor (including inhibition of EMT and the size of the prostate cancer stem cell population), antiresorptive, and antiangiogenic mechanisms.

Transforming growth factor-β (TGF-β) is a crucial player in metastasis of breast cancer cells to bone. We previously reported that bone morphogenetic protein-7 (BMP-7) can counteract the effects of TGF-β in vitro and inhibit the progression and formation of osteotropic MDA-BO2 human breast cancer bone metastasis in vivo. Whether BMP-7 acts directly on the breast cancer cells and/or on the cancer-associated bone stroma has remained elusive. Here, we demonstrated that activin-like kinase receptor 2 (ALK2) is the functional BMP-7 type I receptor in MDA-BO2 cells. Subsequently, to restrict BMP7-induced actions to MDA-BO2 cells we engineered these cells to ectopically express a constitutively active variant of ALK2, denoted caALK2. We demonstrate that caALK2 continuously activates the BMP signaling pathway as measured by Smad phosphorylation, BMP transcriptional reporter activity and target gene responses, and antagonized basal and TGF-β-induced transcriptional reporter activation. In vivo tumor-induced osteolysis and metastatic progression was mitigated in mice inoculated with MDA-BO2 cells overexpressing caALK2 compared to a control cell line. These observations suggest that cell autonomous activation of caALK2 or BMP7 over-expression in breast tumor cells inhibit osteolysis and metastatic progression possibly by interfering with the TGF-β-mediated self-amplifying cycle of bone destruction. Abbreviations ActR-II, Activin receptor type II; ALK, Activin receptor-like kinase; caALK, constitutively active ALK; BMP, bone morphogenetic protein; BMPR-II, BMP receptor type II; BRE, BMPresponsive element; caALK2, constitutively active ALK2; EMT, epithelial mesenchymal transition; GFP, green fluorescent protein; HLH, helix-loop-helix; ID, inhibitor of differentiation/DNA binding; μ-CT, micro-computed tomography; MET, mesenchymal epithelial transition; P-Smad, phosphorylated Smad; I-Smad, inhibitory Smad; Smad, small phenotype and mothers against DPP related protein; TGF-β, transforming growth factor β.

Abstract Metastatic progression of advanced prostate cancer is a major clinical problem. Identifying the cell(s) of origin in prostate cancer and its distant metastases may permit the development of more effective treatment and preventive therapies. In this study, aldehyde dehydrogenase (ALDH) activity was used as a basis to isolate and compare subpopulations of primary human prostate cancer cells and cell lines. ALDH-high prostate cancer cells displayed strongly elevated clonogenicity and migratory behavior in vitro. More strikingly, ALDH-high cells readily formed distant metastases with strongly enhanced tumor progression at both orthotopic and metastatic sites in preclinical models. Several ALDH isoforms were expressed in human prostate cancer cells and clinical specimens of primary prostate tumors with matched bone metastases. Our findings suggest that ALDH-based viable cell sorting can be used to identify and characterize tumor-initiating and, more importantly perhaps, metastasis-initiating cells in human prostate cancer. Cancer Res; 70(12); 5163–73. ©2010 AACR.

Colonisation of bone marrow is a critical step in the formation of bone metastasis. Despite the predominant osteoblastic nature of the bone metastases in prostate cancer, tumour-induced osteoclastic bone destruction is prevalent. Furthermore, angiogenesis is required for the growth of skeletal micro-metastases into clinically overt bone metastases. Bone homing by cancer cells, tumour-induced angiogenesis and osteoclastic bone resorption are dependent on adhesion receptors of the integrin family, particularly αvβ3 and αvβ5 integrins.

A patient with mediastinal liposarcoma causing left ventricular inflow tract obstruction is described. The location of the tumor and its pressure effect were accurately diagnosed by transesophageal echocardiography.

To examine the impact on survival and clinical course of incorporating the morphologic classification of the right ventricle into the evolving management strategy for babies with pulmonary atresia and intact ventricular septum, the surgical results and follow-up status of the first 62 consecutive patients managed in this hospital between 1979 and 1990 were reviewed. Before 1984, all 23 babies from group I underwent primary right ventricular outflow reconstruction irrespective of right ventricular morphology and size. Since 1984, depending on the morphology and size of the right ventricle, 39 babies from group II had either closed transventricular pulmonary valvotomy (n = 31) or a shunt operation (n = 8). There were 10 hospital (43%) and 2 late deaths (total mortality 52%) in our group I patients. Three of the 11 long-term survivors had cyanosis at rest but none had any residual pressure gradient across the pulmonary outflow. Group II had 6 hospital (15%) and 4 late deaths (total mortality = 26%). Of the 29 long-term survivors, 9 had a second-stage right ventricular outflow reconstruction, 8 had balloon valvuloplasty and 2 had successful Fontan operation. At the latest follow-up, 5 children from this group have cyanosis at rest, 1 has a residual gradient (55 mm Hg) across the infundibulum, and 3 have right ventricular dysfunction. The hospital and total mortality for babies in group II was significantly lower than that in group I (p < 0.01). These data suggest that tailoring the treatment to the right ventricular anatomy results in a lower overall mortality although long term postoperative hemodynamic abnormalities are observed in both groups.